Treatment and diagnosis of cancer

ABSTRACT

The present invention is directed to the use of antibodies or binding portions thereof, probes, ligands, or other biological agents which either recognize an extracellular domain of prostate specific membrane antigen or bind to and are internalized with prostate specific membrane antigen. These biological agents can be labeled and used for detection of cancerous tissues, particularly cancerous tissues proximate to or containing vascular endothelial cells, which express an extracellular domain of prostate specific membrane antigen. The labeled biological agents can also be used to detect normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof. They also can be used alone or bound to a substance effective to ablate or kill such cells as a therapy for prostate or other cancers. Also disclosed are four hybridoma cell lines, each of which produces a monoclonal antibody recognizing extracellular domains of prostate specific membrane antigens of normal, benign hyperplastic, and cancerous prostate epithelial cells or portions thereof.

[0001] The present application claims the benefit of U.S. ProvisionalPatent Application 60/022,125, filed Jul. 18, 1996, and is acontinuation-in-part of U.S. patent application Ser. No. 08/838,682,filed Apr. 9, 1997, which claims the benefit of U.S. Provisional PatentApplication Serial No. 60/016,976, filed May 6, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to the treatment and diagnosis ofcancer with biological agents.

BACKGROUND OF THE INVENTION

[0003] In spite of improved treatments for certain forms of cancer, itis still a leading cause of death in the United States. Since the chancefor complete remission of cancer is, in most cases, greatly enhanced byearly diagnosis, it is very desirable that physicians be able to detectcancers before a substantial tumor develops. However, the development ofmethods that permit rapid and accurate detection of many forms ofcancers continues to challenge the medial community. One suchillustrative form of cancer is prostate cancer.

[0004] Prostate cancer is the most common cancer in men with anestimated 317,000 cases in 1996 in the United States. It is the secondleading cause of death among men who die from neoplasia with anestimated 40,000 deaths per year. Prompt detection and treatment isneeded to limit mortality caused by prostate cancer.

[0005] Detection of Prostate Cancer

[0006] When it metastasizes, prostatic cancer has a distinctpredilection for bone and lymph nodes. Saitoh et al., “MetastaticPatterns of Prostatic Cancer. Correlation Between Sites And Number OfOrgans Involved,” Cancer, 54:3078-3084 (1984). At the time of clinicaldiagnosis, as many as 25% of patients have bone metastasis demonstrableby radionuclide scans. Murphy, G. P., et al., “The National Survey OfProstate Cancer In The United States By The American College OfSurgeons,” J. Urol., 127:928-939 (1982). Accurate clinical evaluation ofnodal involvement has proven to be difficult. Imaging techniques such ascomputed tomography (“CT”) or magnetic resonance (“MR”) imaging areunable to distinguish metastatic prostate cancer involvement of lymphnodes by criterion other than size (i.e., >1 cm). Therefore, bydefinition, these imaging modalities are inherently insensitive in thedetection of small volume (<1 cm) disease as well as non-specific in thedetection of larger volume adenopathy. A recent study assessed theaccuracy of MR in patients with clinically localized prostate cancer.Rifkin et al., “Comparison Of Magnetic Resonance Imaging AndUltrasonography In Staging Early Prostate Cancer,” N. Engel. J. Med.,323:621-626 (1990). In this study, 194 patients underwent an MR and 185of these patients had a lymph node dissection. 23 (13%) patients hadpathologically involved lymph nodes. MR was suspicious in only 1 ofthese 23 cases resulting in a sensitivity of 4%. Similar results havealso been noted with CT-scans. Gasser et al., “MRI And UltrasonographyIn Staging Prostate Cancer,” N. Enql. J. Med. (Correspondence),324(7):49-495 (1991).

[0007] The elevation of serum acid phosphatase activity in patientshaving metastasized prostate carcinoma was first reported by Gutman etal., J. Clin Invest 17:473 (1938). In cancer of the prostate, prostaticacid phosphatase is released from the cancer tissue into the bloodstream with the result that the total serum acid phosphatase level canbe greatly increased above normal values. Numerous studies of thisenzyme and its relation to prostatic cancer have been made since thattime, e.g. Yam, Amer. J. Med. 56:604 (1974). However, the measurement ofserum acid phosphatase is elevated in about 65-90 percent of patientshaving carcinoma of the prostate with bone metastasis; in about 30percent of patients without roentgenological evidence of bonemetastasis; and in about only 5-10 percent of patients lackingclinically demonstrable metastasis.

[0008] Prior art attempts to develop a specific test for prostatic acidphosphatase have met with only limited success, because techniques whichrely on enzyme activity on a so-called “specific” substrate cannot takeinto account other biochemical and immunochemical differences among themany acid phosphatases which are unrelated to enzyme activity ofprostate origin. In the case of isoenzymes, i.e. genetically definedenzymes having the same characteristic enzyme activity and a similarmolecular structure but differing in amino acid sequences and/or contentand, therefore, immunochemically distinguishable, it would appearinherently impossible to distinguish different isoenzyme forms merely bythe choice of a particular substrate. It is, therefore, not surprisingthat none of these prior art methods is highly specific for the directdetermination of prostatic acid phosphatase activity; e.g. see Cancer5:236 (1952); J. Lab. Clin. Med. 82:486 (1973); Clin. Chem. Acta. 44:21(1973); and J. Physiol. Chem. 356:1775 (1975).

[0009] In addition to the aforementioned problems of non-specificitywhich appear to be inherent in many of the prior art reagents employedfor the detection of prostate acid phosphatase, there have been reportsof elevated serum acid phosphatase associated with other diseases, whichfurther complicates the problem of obtaining an accurate clinicaldiagnosis of prostatic cancer. For example, Tuchman et al., Am. J. Med.27:959 (1959) noted that serum acid phosphatase levels appear to beelevated in patients with Gaucher's disease.

[0010] Due to the inherent difficulties in developing a “specific”substrate for prostate acid phosphatase, several researchers havedeveloped immunochemical methods for the detection of prostate acidphosphatase. However, the previously reported immunochemical methodshave drawbacks of their own which have precluded their widespreadacceptance. For example, Shulman et al., Immunology 93:474 (1964)described an immuno-diffusion test for the detection of human prostateacid phosphatase. Using antisera prepared from a prostatic fluid antigenobtained by rectal massage from patients with prostatic disease, nocross-reactivity precipitin line was observed in the double diffusiontechnique against extracts of normal kidney, testicle, liver, and lung.However, this method has the disadvantages of limited sensitivity, evenwith the large amounts of antigen employed, and of employing antiserawhich may cross-react with other, antigenically unrelated serum proteincomponents present in prostatic fluid.

[0011] WO 79/00475 to Chu et. al. describes a method for the detectionof prostatic acid phosphatase isoenzyme patterns associated withprostatic cancer which obviates many of the above drawbacks. However,practical problems are posed by the need for a source of cancerousprostate tissue from which the diagnostically relevant prostatic acidphosphatase isoenzyme patterns associated with prostatic cancer areextracted for the preparation of antibodies thereto.

[0012] In recent years, considerable effort has been spent to identifyenzyme or antigen markers for various types of malignancies with theview towards developing specific diagnostic reagents. The ideal tumormarker would exhibit, among other characteristics, tissue or cell-typespecificity. Previous investigators have demonstrated the occurrence ofhuman prostate tissue-specific antigens.

[0013] Treatment of Prostate Cancer

[0014] As described in W. J. Catalona, “Management of Cancer of theProstate,” New Engl. J. Med., 331(15) :996-1004 (1994), the managementof prostate cancer can be achieved by watchful waiting, curativetreatment, and palliation.

[0015] For men with a life expectancy of less than 10 years, watchfulwaiting is appropriate where low-grade, low-stage prostate cancer isdiscovered at the time of a partial prostatectomy for benignhyperplasia. Such cancers rarely progress during the first five yearsafter detection. On the other hand, for younger men, curative treatmentis often more appropriate.

[0016] Where prostate cancer is localized and the patient's lifeexpectancy is 10 years or more, radical prostatectomy offers the bestchance for eradication of the disease. Historically, the drawback ofthis procedure is that most cancers had spread beyond the bounds of theoperation by the time they were detected. However, the use ofprostate-specific antigen testing has permitted early detection ofprostate cancer. As a result, surgery is less extensive with fewercomplications. Patients with bulky, high-grade tumors are less likely tobe successfully treated by radical prostatectomy.

[0017] After surgery, if there are detectable serum prostate-specificantigen concentrations, persistent cancer is indicated. In many cases,prostate-specific antigen concentrations can be reduced by radiationtreatment. However, this concentration often increases again within twoyears.

[0018] Radiation therapy has also been widely used as an alternative toradical prostatectomy. Patients generally treated by radiation therapyare those who are older and less healthy and those with higher-grade,more clinically advanced tumors. Particularly preferred procedures areexternal-beam therapy which involves three dimensional, conformalradiation therapy where the field of radiation is designed to conform tothe volume of tissue treated; interstitial-radiation therapy where seedsof radioactive compounds are implanted using ultrasound guidance; and acombination of external-beam therapy and interstitial-radiation therapy;

[0019] For treatment of patients with locally advanced disease, hormonaltherapy before or following radical prostatectomy or radiation therapyhas been utilized. Hormonal therapy is the main form of treating menwith disseminated prostate cancer. Orchiectomy reduces serumtestosterone concentrations, while estrogen treatment is similarlybeneficial. Diethylstilbestrol from estrogen is another useful hormonaltherapy which has a disadvantage of causing cardiovascular toxicity.When gonadotropin-releasing hormone agonists are administeredtestosterone concentrations are ultimately reduced. Flutamide and othernonsteroidal, anti-androgen agents block binding of testosterone to itsintracellular receptors. As a result, it blocks the effect oftestosterone, increasing serum testosterone concentrations and allowspatients to remain potent—a significant problem after radicalprostatectomy and radiation treatments.

[0020] Cytotoxic chemotherapy is largely ineffective in treatingprostate cancer. Its toxicity makes such therapy unsuitable for elderlypatients. In addition, prostate cancer is relatively resistant tocytotoxic agents.

[0021] Use of Monoclonal Antibodies in Prostate Cancer Detection andTreatment

[0022] Theoretically, radiolabeled monoclonal antibodies (“mAbs”) offerthe potential to enhance both the sensitivity and specificity ofdetecting prostatic cancer within lymph nodes and elsewhere. While manymAbs have previously been prepared against prostate related antigens,none of these mAbs were specifically generated with an imaging objectivein mind. Nevertheless, the clinical need has led to evaluation of someof these mabs as possible imaging agents. Vihko et al., “Radioimaging ofProstatic Carcinoma With Prostatic Acid Phosphatase—SpecificAntibodies,” Biotechnology in Diagnostics, 131-134 (1985); Babaian etal., “Radioimmunological Imaging of Metastatic Prostatic Cancer With111-Indium-Labeled Monoclonal Antibody PAY 276,” J. Urol., 137:439-443(1987); Leroy et al., “Radioimmunodetection Of Lymph Node Invasion InProstatic Cancer. The Use Of Iodine 123 (123-I)-Labeled MonoclonalAnti-Prostatic Acid Phosphatase (PAP) 227 A F (ab′) 2 Antibody FragmentsIn Vivo,” Cancer, 64:1-5 (1989); Meyers et al., “Development OfMonoclonal Antibody Imaging Of Metastatic Prostatic Carcinoma,” TheProstate, 14:209-220 (1989).

[0023] In some cases, the monoclonal antibodies developed for detectionand/or treatment of prostate 20 cancer recognize antigens specific tomalignant prostatic tissues. Such antibodies are thus used todistinguish malignant prostatic tissue (for treatment or detection) frombenign prostatic tissue. See U.S. Pat. No. 4,970,299 to Bazinet et al.and U.S. Pat. No. 4,902,615 to Freeman et al.

[0024] Other monoclonal antibodies react with surface antigens on allprostate epithelial cells whether cancerous or benign. See U.S. Pat.Nos. 4,446,122 and Re 33,405 to Chu et al., U.S. Pat. No. 4,863,851 toMcEwan et al., and U.S. Pat. No. 5,055,404 to Ueda et al. However, theantigens detected by these monoclonal antibodies are present in theblood and, therefore, compete with antigens at tumor sites for themonoclonal antibodies. This causes background noise which makes the useof such antibodies inadequate for in vivo imaging. In therapy, suchantibodies, if bound to a cytotoxic agent, could be harmful to otherorgans.

[0025] Horoszewicz et al., “Monoclonal Antibodies to a New AntigenicMarker in Epithelial Prostatic Cells and Serum of Prostatic CancerPatients,” Anticancer Research, 7:927-936 (1987) (“Horoszewicz”) andU.S. Pat. No. 5,162,504 to Horoszewicz describe an antibody, designated7E11, which recognizes prostate specific membrane antigen (“PSMA”).Israeli et al., “Molecular Cloning of a Complementary DNA Encoding aProstate-specific Membrane Antigen,” Cancer Research, 53:227-230 (1993)(“Israeli”) describes the cloning and sequencing of PSMA and reportsthat PSMA is prostate-specific and shows increased expression levels inmetastatic sites and in hormone-refractory states. Other studies haveindicated that PSMA is more strongly expressed in prostate cancer cellsrelative to -cells from the normal prostate or from a prostate withbenign hyperplasia. Furthermore, PSMA is not found in serum (Troyer etal., “Detection and Characterization of the Prostate-Specific MembraneAntigen (PSMA) in Tissue Extracts and Body Fluids,” Int. J. Cancer,62:552-558 (1995)).

[0026] These characteristics make PSMA an attractive target for antibodymediated targeting for imaging and therapy of prostate cancer. - Imagingstudies using indium-labeled 7E11 have indicated that the antibodylocalizes quite well to both the prostate and to sites of metastasis. Inaddition, 7E11 appears to have clearly improved sensitivity fordetecting lesions compared to other currently available imagingtechniques, such as CT and MR imaging or bone scan. Bander, “CurrentStatus of Monoclonal Antibodies for Imaging and Therapy of ProstateCancer,” Sem. In Oncology, 21:607-612 (1994).

[0027] However, the use of 7E11 and other known antibodies to PSMA tomediate imaging and therapy has several disadvantages. First, PSMA is anintegral membrane protein known to have a short intracellular tail and along extracellular domain. Biochemical characterization and mapping(Troyer et al., “Biochemical Characterization and Mapping of the7E11-C5.3 Epitope of the Prostate-specific Membrane Antigen,” Urol.Oncol., 1:29-37 (1995)) have shown that the epitope or antigenic site towhich the 7E11 antibody binds is present on the intracellular portion ofthe molecule. Because antibody molecules do not, under normalcircumstances, cross the cell membrane unless they bind to theextracellular portion of a molecule and become translocatedintracellularly, the 7E11 antibody does not have access to its antigenictarget site in an otherwise healthy, viable cell.

[0028] Consequently, imaging using 7E11 is limited to the detection ofdead cells within tumor deposits. Additionally, the therapeutic use ofthe 7E11 antibody is limited, because only cells that are already deador tissue containing a large proportion of dead cells can be effectivelytargeted.

[0029] Although the inadequacies and problems in the diagnosis andtreatment of one particular type of cancer are the focus of thepreceding discussion, prostate cancer is merely a representative model.The diagnosis and treatment of numerous other cancers have similarproblems.

[0030] The present invention is directed to overcoming the deficienciesof prior art antibodies in diagnosing and treating prostate and othertypes of cancer.

SUMMARY OF THE INVENTION

[0031] One aspect of the present invention relates to a method ofablating or killing cancerous cells. The process involves providing abiological agent which, when contacted with an extracellular domain ofprostate specific membrane antigen, recognizes the extracellular domainof prostate specific membrane antigen. These biological agents arecontacted with vascular endothelial cells proximate to the cancerouscells under conditions effective to permit both binding of thebiological agent to the vascular endothelial cells proximate to thecancerous cells and killing or ablating of the cancerous cells. Thebiological agent can be used alone or can be bound to a substanceeffective to kill or ablate the cancerous cells upon binding of thebiological agent to vascular endothelial cells that are proximate to thecancerous cells.

[0032] In a particularly preferred embodiment of the method of ablatingor killing cancerous cells in accordance with the present invention, thebiological agent, when contacted with an extracellular domain ofprostate specific membrane antigen, binds to and is internalized withthe prostate specific membrane antigen of such cells. Preferredbiological agents for use in the method of ablating or killing cancerouscells in accordance with the present invention are antibodies or bindingportions thereof, probes, or ligands. The methods of the presentinvention are particularly useful in killing or ablating renal,urothelial, colon, rectal, lung, and breast cancerous cells andcancerous cells of metastatic adenocarcinoma to the liver.

[0033] Another aspect of- the present invention relates to a method ofdetecting cancerous tissue in a biological sample. This method involvesproviding a biological agent which, when contacted with an extracellulardomain of prostate specific membrane antigen, binds to the extracellulardomain of prostate specific membrane antigen. The biological agent isbound to a label effective to permit detection of vascular endothelialcells proximate to or within the cancerous tissue upon binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue. The biological sample is contacted with thebiological agent having a label under conditions effective to permitbinding of the biological agent to the vascular endothelial cellsproximate to or within the cancerous tissue in the biological sample.The presence of cancerous tissue in the biological sample is detected bydetection of the label.

[0034] In a particularly preferred embodiment of the method of detectingcancerous tissue in accordance with the present invention, thebiological agent is one that, when contacted with an extracellulardomain of prostate specific membrane antigen, binds to and isinternalized with the prostate specific membrane antigen. Preferredbiological agents for use in the method of detecting cancerous tissue inaccordance with the present invention are antibodies or binding portionsthereof, probes, or ligands. The method is especially useful indetecting renal, urothelial, colon, rectal, lung, and breast canceroustissue and cancerous tissue of metastatic adenocarcinoma to the liver.

[0035] Still another aspect of the present invention relates to a methodof ablating or killing normal, benign hyperplastic, and cancerousprostate epithelial cells. The process involves providing a biologicalagent which recognizes an extracellular domain of prostate specificmembrane antigen. The biological agent can be used alone or can be boundto a substance effective to kill the cells upon binding of thebiological agent to the cells. These biological agents are thencontacted with the cells under conditions effective to permit bothbinding of the biological agent to the extracellular domain of theprostate specific membrane antigen and killing or ablating of the cells.

[0036] In a particularly preferred embodiment of the method of ablatingor killing normal, benign hyperplastic, and cancerous prostateepithelial cells in accordance with the present invention, thebiological agent binds to and is internalized with the prostate specificmembrane antigen of such cells. Preferred biological agents for use inthe method of ablating or killing normal, benign hyperplastic, andcancerous prostate epithelial cells in accordance with the presentinvention are antibodies or binding portions thereof, probes, orligands.

[0037] Another aspect of the present invention relates to a method ofdetecting normal, benign hyperplastic, and cancerous prostate epithelialcells or portions thereof in a biological sample. This method involvesproviding a biological agent which binds to an extratellular domain ofprostate specific membrane antigen. The biological agent is bound to alabel effective to permit detection of the cells or portions thereofupon binding of the biological agent to the cells or portions thereof.The biological sample is contacted with the biological agent having alabel under conditions effective to permit binding of the biologicalagent to the extracellular domain of the prostate specific membraneantigen of any of the cells or portions thereof in the biologicalsample. The presence of any cells or portions thereof in the biologicalsample is detected by detection of the label.

[0038] In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent binds to andis internalized with the prostate specific membrane antigen of suchcells. Preferred biological agents for use in the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention are antibodies or binding portionsthereof, probes, or ligands.

[0039] Another aspect of the present invention pertains to a biologicalagent that recognizes an extracellular domain of prostate specificmembrane antigen. In a preferred embodiment, the isolated biologicalagent binds to and is internalized with the prostate specific membraneantigen. Preferred isolated biological agents which recognize anextracellular domain of prostate specific membrane antigen in accordancewith the present invention are isolated antibodies or binding portionsthereof, probes, or ligands. Hybridoma cell lines that producemonoclonal antibodies of these types are also disclosed.

[0040] The biological agents of the present invention recognize theextracellular domain of antigens of normal, benign hyperplastic, andcancerous prostate epithelial cells. Unlike the 7E11 antibody, whichrecognizes an epitope of prostate-associated antigens which are exposedextracellularly only after cell lysis, the biological agents of thepresent invention bind to antigenic epitopes which are extracellularlyexposed in living prostate cells. Using the biological agents of thepresent invention, living, unfixed normal, benign hyperplastic, andcancerous prostate epithelial cells can be targeted, which makestreatment and diagnosis more effective. In a preferred embodiment fortreating prostate cancer, the biological agents of the present inventionalso bind to and are internalized with the prostate specific membraneantigen, which permits the therapeutic use of intracellularly actingcytotoxic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is an immuno-electron micrograph of gold-labeled monoclonalantibody J591 on the surface of LNCaP. cells after incubation at 4° C.

[0042]FIG. 2 is an immuno-electron micrograph of LNCaP cells treatedwith gold-labeled monoclonal antibody J591 after 5 minutes incubation at37° C.

[0043]FIG. 3 is an immuno-electron micrograph of LNCaP cells treatedwith gold-labeled monoclonal antibody J591 after 10 minutes incubationat 37° C.

[0044]FIG. 4 is an immuno-electron micrograph of LNCaP cells treatedwith gold-labeled monoclonal antibody J591 after 15 minutes incubationat 37° C.

[0045]FIG. 5 is an immuno-electron micrograph of LNCaP cells treatedwith gold-labeled monoclonal antibody J591 after 15 minutes at 37° C.showing J591 within endosomes.

[0046]FIG. 6 summarizes the sequencing strategy of the heavy chain ofmonoclonal antibody J591.

[0047]FIG. 7 shows the nucleotide sequence of the heavy chain ofmonoclonal antibody J591 (designated SEQ.ID. No. 1), the nucleotidesequence of the corresponding reverse, non-coding strand (designatedSEQ. ID. No. 2), and the corresponding deduced amino acid sequences(designated SEQ. ID. Nos. 3, 4, and 5).

[0048]FIG. 8 is a comparison of the heavy chain of monoclonal antibodyJ591 with the consensus sequence for Mouse Heavy Chains Subgroup IIA.

[0049]FIG. 9 summarizes the sequencing strategy of the kappa light chainof monoclonal antibody J591.

[0050]FIG. 10 shows the nucleotide sequences of the kappa light chain ofmonoclonal antibody J591 (designated SEQ.ID. No. 9), the nucleotidesequence of the corresponding reverse, non-coding strand (designatedSEQ. ID. No. 10), and the corresponding deduced amino acid sequence(designated SEQ. ID. Nos. 11, 12, and 13).

[0051]FIG. 11 is a comparison of the kappa light chain of monoclonalantibody J591 with the consensus sequence for Mouse Kappa ChainsSubgroup V.

[0052] FIGS. 12A-12F are micrographs (250× magnification) showing theimmunohistochemical reactivity of mAb J591 to neovasculature of variouscarcinomas.

DETAILED DESCRIPTION OF THE INVENTION

[0053] One aspect of the present invention relates to a method ofablating or killing normal, benign hyperplastic, and cancerous prostateepithelial cells. the process involves providing a biological agent,such as an antibody or binding portion thereof, probe, or ligand, whichbinds to an extracellular domain of prostate specific membrane antigenof (i.e., a portion of prostate specific membrane antigen which isexternal to) such cells. The biological agent can be used alone or canbe bound to a substance effective to kill the cells upon binding of thebiological agent to the cells. These biological agents are thencontacted with the cells under conditions effective to permit bothbinding of the biological agent to the extracellular domain of theprostate specific membrane antigen and killing or ablating of the cells.In its preferred form, such contacting is carried out in a living mammalby administering the biological agent to the mammal under conditionseffective to permit both binding of the biological agent to theextracellular domain of the prostate specific membrane antigen andkilling or ablating of the cells. Such administration can be carried outorally or parenterally.

[0054] In a particularly preferred embodiment of the method of ablatingor killing normal, benign hyperplastic, and cancerous prostateepithelial cells in accordance with the present invention, thebiological agent binds to and is internalized with the prostate specificmembrane antigen of such cells. Again, the biological agent can be usedalone. Alternatively, the biological agent can be bound to a substanceeffective to kill the cells upon binding of the biological agent toprostate specific membrane antigen and upon internalization of thebiological agent with the prostate specific membrane antigen.

[0055] The mechanism by which the biological agent is internalized withthe prostate specific membrane antigen is not critical to the practiceof the present invention. For example, the biological agent can induceinternalization of the prostate specific membrane antigen.Alternatively, internalization of the biological agent can be the resultof routine internalization of prostate specific membrane antigen.

[0056] The above-described biological agents (i.e., biological agents,such as an antibody or binding portion thereof, probe, or ligand which,when contacted with an extracellular domain of prostate specificmembrane antigen, recognizes the extracellular domain of prostatespecific membrane antigen and, preferably, is internalized therewith)can be used to ablate or kill cancerous cells. In this aspect of thepresent invention, the biological agent can be used alone or can bebound to a substance effective to kill the cancerous cells upon bindingof the biological agent to vascular endothelial cells proximate thereto.These biological agents are contacted with vascular endothelial cellsproximate to the cancerous cells. The contacting is carried out underconditions that are effective to permit binding of the biological agentto the vascular endothelial cells proximate to the cancerous cells and,in addition, that are effective to kill or ablate the cancerous cells.The mechanism by which the cancerous cells are killed or ablated is notcritical to the practice of the present invention. For example, thecancerous cells can be killed or ablated directly by the biologicalagent as a consequence of their proximity to the vascular endothelialcells to which the biological agent binds. Alternatively, the biologicalagent can kill, ablate, or otherwise change the properties of thevascular endothelial cells to which it binds so that blood flow to thecancerous cells proximate thereto is stopped or otherwise reduced,thereby causing the cancerous cells to be killed or ablated. Thus, themethod of the present invention is particularly useful for killing orablating vascular endothelial cells in cancerous tissue as well as thecancerous cells contained in cancerous tissue.

[0057] In a particularly preferred embodiment of the method of ablatingor killing cancerous cells in accordance with the present invention, thebiological agent employed is one that, when contacted with anextracellular domain of prostate specific membrane antigen, binds to andis internalized with the extracellular domain of prostate specificmembrane antigen. The methods of the present invention are particularlyuseful to kill or ablate cancerous prostate epithelial cells as well ascancerous cells other than cancerous prostate epithelial cells. Examplesof cancerous cells which are not cancerous prostate epithelial cells arerenal, urothelial, colon, rectal, lung, and breast cancerous cells andcancerous cells of metastatic adenocarcinoma to the liver. Although themethod of the present invention can be used to kill or ablate any cellwhich expresses an extracellular domain of prostate specific membraneantigen or a portion thereof or whose subsistence is dependent uponcells which express an extracellular domain of prostate specificmembrane antigen or a portion thereof, the method of the presentinvention is particularly useful to kill or ablate cancerous cells,because the vascular endothelial cells supplying blood to canceroustissues (e.g., tumors, collections of cancerous cells, or othercancerous masses) express an extracellular domain of prostate specificmembrane antigen, irrespective of the type of cancer involved. Incontrast, vascular endothelial cells supplying blood to normal tissuesdo not express an extracellular domain of prostate specific membraneantigen.

[0058] Another aspect of the present invention relates to a method ofdetecting normal, benign hyperplastic, and cancerous epithelial cells orportions thereof in a biological sample. This method involves providinga biological agent, such as an antibody or binding portion thereof,probe, or ligand, which binds to an extracellular domain of prostatespecific membrane antigen of such cells. The biological agent is boundto a label effective to permit detection of the cells or portions (e.g.,prostate specific membrane antigen or fragments thereof liberated fromsuch normal, benign hyperplastic, and cancerous cells) thereof uponbinding of the biological agent to the cells or portions thereof. Thebiological sample is contacted with the biological agent having a labelunder conditions effective to permit binding of the biological agent tothe extracellular domain of the prostate specific membrane antigen ofany of the cells or portions thereof in the biological sample. Thepresence of any cells or portions thereof in the biological sample isdetected by detection of the label. In its preferred form, suchcontacting is carried out in a living mammal and involves administeringthe biological agent to the mammal under conditions effective to permitbinding of the biological agent to the prostate specific membraneantigen of any of the cells or portions thereof in the biologicalsample. Again, such administration can be carried out orally orparenterally.

[0059] The method of the present invention can be used to screenpatients for diseases associated with the presence of normal, benignhyperplastic, and cancerous epithelial cells or portions thereof.Alternatively, it can be used to identify the recurrence of suchdiseases, particularly when the disease is localized in a particularbiological material of the patient. For example, recurrence of prostaticdisease in the prostatic fossa may be encountered following radicalprostatectomy. Using the method of the present invention, thisrecurrence can be detected by administering a short range radiolabeledantibody to the mammal and then detecting the label rectally, such aswith a transrectal detector probe.

[0060] Alternatively, the contacting step can be carried out in a sampleof serum or urine or other body fluids, such as to detect the presenceof PSMA in the body fluid. When the contacting is carried out in a serumor urine sample, it is preferred that the biological agent recognizesubstantially no antigens circulating in the blood other than PSMA.Since intact prostate cells do not excrete or secrete PSMA into theextracellular environment, detecting PSMA in serum, urine, or other bodyfluids generally indicates that prostate cells are being lysed. Thus,the biological agents and methods of the present invention can be usedto determine the effectiveness of a prostate cancer treatment protocolby monitoring the level of PSMA in serum, urine or other body fluids.

[0061] In a particularly preferred embodiment of the method of detectingnormal, benign hyperplastic, and cancerous prostate epithelial cells inaccordance with the present invention, the biological agent, such as theantibody or binding portion thereof, probe, or ligand, binds to and isinternalized with the prostate specific membrane antigen.. of suchcells. Again, the biological agent is bound to a label effective topermit detection of the cells or portions thereof upon binding of thebiological agent to and internalization of the biological agent with theprostate specific membrane antigen.

[0062] Another aspect of the present invention relates to a method ofdetecting cancerous tissue in a biological sample. This method involvesproviding the above-described biological agent (i.e., a biologicalagent, such as an antibody or binding portion thereof, probe, or ligandwhich, when contacted with an extracellular domain of prostate specificmembrane antigen, recognizes the extracellular domain of prostatespecific membrane antigen). The biological agent is bound to a labelthat is effective to permit detection of vascular endothelial cellsproximate to or within the cancerous tissue upon binding of thebiological agent to vascular endothelial cells proximate to or withinthe cancerous tissue. The biological sample is then contacted with thebiological agent having a label. Contacting is carried out underconditions effective to permit binding of the biological agent to thevascular endothelial cells proximate to or within the cancerous tissuein the biological sample. The presence of cancerous cells or portionsthereof in the biological sample is detected by detection of the label.

[0063] Rather than contacting the entire biological sample with thebiological agent, it is contemplated that a portion of the biologicalsample can be used. For example, a tissue biopsy sample can be contactedwith the biological agent to determine the presence of cancerous tissuein the tissue biopsy sample as well as in the larger biological samplefrom which it is taken. Alternatively, the biological agent can becontacted with a serum or urine sample to acertain whether any vascularendothelial cells expressing an extracellular domain of prostatespecific membrane antigen are present therein. Since vascularendothelial cells expressing an extracellular domain of prostatespecific membrane antigen are found in the vasculature of canceroustissues but not in the vasculature of normal tissues, detection of thelabel in a serum or urine sample indicates the presence of canceroustissue in the larger biological sample from which it is taken (e.g., apatient).

[0064] In a particularly preferred embodiment of the method of detectingcancerous tissues in accordance with the present invention, thebiological agent employed is one that, when contacted with anextracellular domain of prostate specific membrane antigen, binds to andis internalized with the prostate specific membrane antigen. The methodsof the present invention can be used to detect cancerous prostateepithelial cells as well as cancerous tissues containing cancerous cellsother than cancerous prostate epithelial cells. Examples of canceroustissues containing cancerous cells other than cancerous prostateepithelial cells which can be detected with the methods of the presentinvention include renal, urothelial, colon, rectal, lung, and breastcancerous tissue and cancerous tissue of metastatic adenocarcinoma tothe liver.

[0065] As indicated above, biological agents suitable for eitherkilling, ablating, or detecting cancerous cells and normal, benignhyperplastic, and cancerous prostate epithelial cells includeantibodies, such as monoclonal or polyclonal antibodies. In addition,antibody fragments, half-antibodies, hybrid derivatives, probes, andother molecular constructs may be utilized. These biological agents,such as antibodies, binding portions thereof, probes, or ligands, bindto extracellular domains of prostate specific membrane antigens orportions thereof in normal, benign hyperplastic, and cancerous prostateepithelial cells. As a result, when practicing the methods of thepresent invention to kill, ablate, or detect normal, benignhyperplastic, and cancerous prostate epithelial cells, the biologicalagents bind to all such cells, not only to cells which are fixed orcells whose intracellular antigenic domains are otherwise exposed to theextracellular environment. Consequently, binding of the biologicalagents is concentrated in areas where there are prostate epithelialcells, irrespective of whether these cells are fixed or unfixed, viableor necrotic. Additionally or alternatively, these biological agents,such as antibodies, binding portions thereof, probes, or ligands, bindto and are internalized with prostate specific membrane antigens orportions thereof in normal, benign hyperplastic, and cancerous prostateepithelial cells.

[0066] Monoclonal antibody production may be effected by techniqueswhich are well-known in the art. Basically, the process involves firstobtaining immune cells (lymphocytes) from the spleen of a mammal (e.g.,mouse) which has been previously immunized with the antigen of interesteither in vivo or in vitro. The antibody-secreting lymphocytes are thenfused with (mouse) myeloma cells or transformed cells, which are capableof replicating indefinitely in cell culture, thereby producing animmortal, immunoglobulin-secreting cell line. The resulting fused cells,or hybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned, and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature 256:495 (1975), which is hereby incorporated byreference.

[0067] Mammalian lymphocytes are immunized by in vivo immunization ofthe animal (e.g., a mouse) with the protein or polypeptide of thepresent invention. Such immunizations are repeated as necessary atintervals of up to several weeks to obtain a sufficient titer ofantibodies. Following the last antigen boost, the animals are sacrificedand spleen cells removed.

[0068] Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is effected bystandard and well-known techniques, for example, by using polyethyleneglycol (“PEG”) or other fusing agents (See Milstein and Kohler, Eur. J.Immunol. 6:511 (1976), which is hereby incorporated by reference). Thisimmortal cell line, which is preferably murine, but may also be derivedfrom cells of other mammalian species, including but not limited to ratsand humans, is selected to be deficient in enzymes necessary for theutilization of certain nutrients, to be capable of rapid growth, and tohave good fusion capability. Many such cell lines are known to thoseskilled in the art, and others are regularly described.

[0069] Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain syntheticsurfactant adjuvant pluronic polyols, or pulverized acrylamide gelcontaining the protein or polypeptide after SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. Ultimately, the rabbits are euthenized with pentobarbital 150mg/Kg IV. This and other procedures for raising polyclonal antibodiesare disclosed in E. Harlow, et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference.

[0070] In addition to utilizing whole antibodies, the processes of thepresent invention encompass use of binding portions of such antibodies.Such binding portions include Fab fragments, F(ab′)₂ fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118(N.Y. Academic Press 1983), which is hereby incorporated by reference.

[0071] Alternatively, the processes of the present invention can utilizeprobes or ligands found either in nature or prepared synthetically byrecombinant DNA procedures or other biological or molecular procedures.Suitable probes or ligands are molecules which bind to the extracellulardomains of prostate specific membrane antigens identified by themonoclonal antibodies of the present invention. Other suitable probes orligands are molecules which bind to and are internalized with prostatespecific membrane antigens. Such probes or ligands can be, for example,proteins, peptides, lectins, or nucleic acid probes.

[0072] It is particularly preferred to use the monoclonal antibodiesidentified below in Table 1. TABLE 1 Monoclonal ATCC Designation forAntibody Name Hybridoma Cell Line E99 HB-12101 J415 HB-12109 J533HB-12127 J591 HB-12126

[0073] These antibodies can be used alone or as a component in a mixturewith other antibodies or other biological agents to treat cancers orimage cancerous tissues (particularly the vascular endothelial cellstherein) or prostate epithelial cells with varying surface antigencharacteristics.

[0074] Regardless of whether the biological agents are used fortreatment or diagnosis, they can be administered orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes, such as, that of the nose, throat, and bronchialtubes. They may be administered alone or with pharmaceutically orphysiologically acceptable carriers, excipients, or stabilizers, and canbe in solid or liquid form such as, tablets, capsules, powders,solutions, suspensions, or emulsions.

[0075] The solid unit dosage forms can be of the conventional type. Thesolid form can be a capsule, such as an ordinary gelatin type containingthe biological agent, such as an antibody or binding portion thereof, ofthe present invention and a carrier, for example, lubricants and inertfillers such as, lactose, sucrose, or cornstarch. In another embodiment,these compounds are tableted with conventional tablet bases such aslactose, sucrose, or cornstarch in combination with binders like acacia,cornstarch, or gelatin, disintegrating agents such as, cornstarch,potato starch, or alginic acid, and a lubricant like stearic acid ormagnesium stearate.

[0076] The biological agent of the present invention may also beadministered in injectable dosages by solution or suspension of thesematerials in a physiologically acceptable diluent with a pharmaceuticalcarrier. Such carriers include sterile liquids such as water and oils,with or without the addition of a surf actant and other pharmaceuticallyand physiologically acceptable carrier, including adjuvants, excipientsor stabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions.

[0077] For use as aerosols, the biological agent of the presentinvention in solution or suspension may be packaged in a pressurizedaerosol container together with suitable propellants, for example,hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

[0078] The biological agents may be utilized to detect cancerous tissues(particularly the vascular endothelial cells therein) and normal, benignhyperplastic, and cancerous prostate epithelial cells in vivo. This isachieved by labeling the biological agent, administering the labeledbiological agent to a mammal, and then imaging the mammal.

[0079] Examples of labels useful for diagnostic imaging in accordancewith the present invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I,⁹⁹mTc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such asfluorescein and rhodamine, nuclear magnetic resonance active labels,positron emitting isotopes detectable by a positron emission tomography(“PET”) scanner, chemiluminescers such as luciferin, and enzymaticmarkers such as peroxidase or phosphatase. Short-range radiationemitters, such as isotopes detectable by short-range detector probes,such as a transrectal probe, can also be employed. These isotopes andtransrectal detector probes, when used in combination, are especiallyuseful in detecting prostatic fossa recurrences and pelvic nodaldisease. The biological agent can be labeled with such reagents usingtechniques known in the art. For example, see Wensel and Meares,Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983),which is hereby incorporated by reference, for techniques relating tothe radiolabeling of antibodies. See also, D. Colcher et al., “Use ofMonoclonal Antibodies as Radiopharmaceuticals for the Localization ofHuman Carcinoma Xenografts in Athymic Mice”, Meth. Enzymol. 121: 802-816(1986), which is hereby incorporated by reference.

[0080] A radiolabeled biological agent of this invention can be used forin vitro diagnostic tests. The specific activity of a tagged biologicalagent, such as a tagged antibody, binding portion thereof, probe, orligand, depends upon the half-life, the isotopic purity of theradioactive label, and how the label is incorporated into the biologicalagent. Table 2 lists several commonly-used isotopes, their specificactivities and half-lives. In immunoassay tests, the higher the specificactivity, in general, the better the sensitivity. TABLE 2 SpecificActivity of Pure Isotope Isotope (Curies/mole) Half-Life ¹⁴C 6.25 × 10¹5720 years ³H 2.01 × 10⁴ 12.5 years ³⁵S 1.50 × 10⁶ 87 days ¹²⁵I 2.18 ×10⁶ 60 days ³²P 3.16 × 10⁶ 14.3 days ¹³¹I 1.62 × 10⁷ 8.1 days

[0081] Procedures for labeling biological agents with the radioactiveisotopes listed in Table 2 are generally known in the art. Tritiumlabeling procedures are described in U.S. Pat. No. 4,302,438, which ishereby incorporated by reference. Iodinating, tritium labeling, and 3Slabeling procedures especially adapted for murine monoclonal antibodiesare described by Goding, J. W. (supra, pp 124-126) and the referencescited therein, which are hereby incorporated by reference. Otherprocedures for iodinating biological agents, such as antibodies, bindingportions thereof, probes, or ligands, are described by Hunter andGreenwood, Nature 144:945 (1962), David et al., Biochemistry13:1014-1021 (1974), and U.S. Pat. Nos. 3,867,517 and 4,376,110, whichare hereby incorporated by reference. Radiolabeling elements which areuseful in imaging include ¹²³I, ¹³¹I, ¹¹¹In, and ^(99m)Tc, for example.Procedures for iodinating biological agents are described by Greenwood,F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J., Biochem. J.113:299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297(1971), which are hereby incorporated by reference. Procedures for^(99m)Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. etal. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,New York: Masson 111-123 (1982) and the references cited therein, whichare hereby incorporated by reference. Procedures suitable for¹¹¹In-labeling biological agents are described by Hnatowich, D.J. etal., J. Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J.Applied Radiation, 35:554-557 (1984), and Buckley, R. G. et al.,F.E.B.S. 166:202-204 (1984), which are hereby incorporated by reference.

[0082] In the case of a radiolabeled biological agent, the biologicalagent is administered to the patient, is localized to the tumor bearingthe antigen with which the biological agent reacts, and is detected or“imaged” in vivo using known techniques such as radionuclear scanningusing e.g., a gamma camera or emission tomography. See e.g., A. R.Bradwell et al., “Developments in Antibody Imaging”, MonoclonalAntibodies for Cancer Detection and Therapy, R. W. Baldwin et al.,(eds.), pp. 65-85 (Academic Press 1985), which is hereby incorporated byreference. Alternatively, a positron emission transaxial tomographyscanner, such as designated Pet VI located at Brookhaven NationalLaboratory, can be used where the radiolabel emits positrons (e.g., ¹¹C,¹⁸F, ¹⁵O, and ¹³N).

[0083] Fluorophore and chromophore labeled biological agents can beprepared from standard moieties known in the art. Since antibodies andother proteins absorb light having wavelengths up to about 310 nm, thefluorescent moieties should be selected to have substantial absorptionat wavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluorescers and chromophores are described by Stryer, Science,162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry,41:843-868 (1972), which are hereby incorporated by reference. Thebiological agents an be labeled with fluorescent chromophore groups byconventional procedures such as those disclosed in U.S. Pat. Nos.3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated byreference.

[0084] One group of fluorescers having a number of the desirableproperties described above are the xanthene dyes, which include thefluoresceins derived from 3,6-dihydroxy-9-henylxanthhydrol and resaminesand rhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

[0085] Biological agents can be labeled with fluorchromes orchromophores by the procedures described by Goding, J. (supra, pp208-249). The biological agents can be labeled with an indicating groupcontaining the NMR-active ¹⁹F atom, or a plurality of such atomsinasmuch as (i) substantially all of naturally abundant fluorine atomsare the ¹⁹F isotope and, thus, substantially all fluorine-containingcompounds are NMR-active; (ii) many chemically active polyfluorinatedcompounds such as trifluoracetic anhydride are commercially available atrelatively low cost, and (iii) many fluorinated compounds have beenfound medically acceptable for use in humans such as the perfluorinatedpolyethers utilized to carry oxygen as hemoglobin replacements. Afterpermitting such time for incubation, a whole body NMR determination iscarried out using an apparatus such as one of those described by Pykett,Scientific American, 246:78-88 (1982), which is hereby incorporated byreference, to locate and image cancerous tissues (particularly thevascular endothelial cells therein) and prostate epithelial cells.

[0086] In cases where it is important to distinguish between regionscontaining live and dead prostate epithelial cells or to distinguishbetween live and dead prostate epithelial cells, the antibodies of thepresent invention (or other biological agents of the present invention),labeled as described above, can be coadministered along with an antibodyor other biological agent which recognizes only living or only deadprostate epithelial cells labeled with a label which can bedistinguished from the label used to label the subject antibody. Bymonitoring the concentration of the two labels at various locations ortimes, spatial and temporal concentration variations of living and deadnormal, benign hyperplastic, and cancerous prostate epithelial cells canbe ascertained. In particular, this method can be carried out using thelabeled antibodies of the present invention, which recognize both livingand dead epithelial prostate cells, and labeled 7E11 antibodies, whichrecognize only dead epithelial prostate cells.

[0087] The biological agents can also be utilized to kill or ablatecancerous cells and normal, benign hyperplastic, and cancerous prostateepithelial cells in vivo. This involves using the biological agents bythemselves or with a cytotoxic drug to which the biological agents ofthe present invention (i.e., biological agents recognizing normal,benign hyperplastic, and cancerous prostate epithelial cells) are bound.This involves administering the biological agents bonded to a cytotoxicdrug to a mammal requiring such treatment. In the case of normal, benignhyperplastic, and cancerous prostate epithelial cells, since thebiological agents recognize prostate epithelial cells, any such cells towhich the biological agents bind are destroyed. Although suchadministration may destroy normal prostate epithelial cells, this is notproblematic, because the prostate is not required for life or survival.Although the prostate may indirectly contribute to fertility, this isnot likely to be a practical consideration in patients receiving thetreatment of the present invention. In the case of cancerous tissues,since the biological agents recognize vascular endothelial cells thatare proximate to cancerous cells, binding of the biologicalagent/cytotoxic drug complex to these vascular endothelial cellsdestroys them, thereby cutting off the blood flow to the proximatecancerous cells and, thus, killing or ablating these cancerous cells.Alternatively, the biological agents, by virtue of their binding tovascular endothelial cells that are proximate to cancerous cells, arelocalized proximate to the cancerous cells. Thus, by use of suitablebiological agents (including those containing substances effective tokill cells nondiscriminatingly but only over a short range), cells incancerous tissue (including cancerous cells) can be selectively killedor ablated.

[0088] The biological agents of the present invention may be used todeliver a variety of cytotoxic drugs including therapeutic drugs, acompound emitting radiation, molecules of plants, fungal, or bacterialorigin, biological proteins, and mixtures thereof. The cytotoxic drugscan be intracellularly acting cytotoxic drugs, such as short-rangeradiation emitters, including, for example, short-range, high-energyβ-emitters.

[0089] Enzymatically active toxins and fragments. thereof areexemplified by diphtheria toxin A fragment, nonbinding active fragmentsof diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin Achain, abrin A chain, modeccin A chain, α-sacrin, certain Aleuritesfordii proteins, certain Dianthin proteins, Phytolacca americanaproteins (PAP, PAPII and PAP-S) , Morodica charantia inhibitor, curcin,crotin, Saponaria officinalis inhibitor, gelonin, mitogillin,restrictocin, phenomycin, and enomycin, for example. Procedures forpreparing enzymatically active polypeptides of the immunotoxins aredescribed in WO84/03508 and WO85/03508, which are hereby incorporated byreference. Certain cytotoxic moieties are derived from adriamycin,chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum,for example.

[0090] Procedures for conjugating the biological agents with thecytotoxic agents have been previously described. Procedures forconjugating chlorambucil with antibodies are described by Flechner, I,.European Journal of Cancer, 9:741-745 (1973); Ghose, T. et al., BritishMedical Journal, 3:495-499 (1972); and Szekerke, M., et al., Neoplasma,19:211-215 (1972), which are hereby incorporated by reference.Procedures for conjugating daunomycin and adriamycin to antibodies aredescribed by Hurwitz, E. et al., Cancer Research, 35:1175-1181 (1975)and Arnon, R. et al. Cancer Surveys, 20 1:429-449 (1982), which arehereby incorporated by reference. Procedures for preparingantibody-ricin conjugates are described in U.S. Pat. No. 4,414,148 andby Osawa, T., et al. Cancer Surveys, 1:373-388 (1982) and the referencescited therein, which are hereby incorporated by reference. Couplingprocedures as also described in EP 86309516.2, which is herebyincorporated by reference.

[0091] In a particularly preferred embodiment of the present invention,especially well-suited for killing or ablating normal, benignhyperplastic, and cancerous prostate epithelial cells, a firstbiological agent is conjugated with a prodrug which is activated onlywhen in close proximity with a prodrug activator. The prodrug activatoris conjugated with a second biological agent according to the presentinvention, preferably one which binds to a non-competing site on theprostate specific membrane antigen molecule. Whether two biologicalagents bind to competing or non-competing binding sites can bedetermined by conventional competitive binding assays. For example,monoclonal antibodies J591, J533, and E99 bind to competing bindingsites on the prostate specific membrane antigen molecule. Monoclonalantibody J415, on the other hand, binds to a binding site which isnon-competing with the site to which J591, J533, and E99 bind. Thus, forexample, the first biological agent can be one of J591, J533, and E99,and the second biological agent can be J415. Alternatively, the firstbiological agent can be J415, and the second biological agent can be oneof J591, J533, and E99. Drug-prodrug pairs suitable for use in thepractice of the present invention are described in Blakely et al.,“ZD2767, an Improved System for Antibody-directed Enzyme Prodrug TherapyThat Results in Tumor Regressions in Colorectal Tumor Xenografts,”Cancer Research, 56:3287-3292 (1996), which is hereby incorporated byreference.

[0092] Alternatively, the biological agent can be coupled to high energyradiation emitters, for example, a radioisotope, such as ¹³¹I, aγ-emitter, which, when localized at the tumor site, results in a killingof several cell diameters. See, e.g., S. E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985),which is hereby incorporated by reference. Other suitable radioisotopesinclude α-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters,such as ¹⁸⁶Re and ⁹⁰Y. Radiotherapy is expected to be particularlyeffective, because prostate epithelial cells and vascular endothelialcells within cancers are relatively radiosensitive.

[0093] Where the biological agents are used alone to kill or ablatecancerous cells or prostate epithelial cells, such killing or ablationcan be effected by initiating endogenous host immune functions, such ascomplement-mediated or antibody-dependent cellular cytotoxicity.

[0094] The biological agent of the present invention can be used andsold together with equipment, as a kit, to detect the particular label.

[0095] Biological agents of the present invention can be used inconjunction with other therapeutic treatment modalities. Such othertreatments include surgery, radiation, cryosurgery, thermotherapy,hormone treatment, chemotherapy, vaccines, and other immunotherapies.

[0096] Also encompassed by the present invention is a method of killingor ablating which involves using the biological agents for prophylaxis.For example, these materials can be used to prevent or delay developmentor progression of prostate or other cancers.

[0097] Use of the therapeutic methods of the present invention to treatprostate and other cancers has a number of benefits. Since thebiological agents according to the present invention only targetcancerous cells (such as cells of cancerous tissues containing vascularendothelial cells) and prostate epithelial cells, other tissue isspared. As a result, treatment with such biological agents is safer,particularly for elderly patients. Treatment according to the presentinvention is expected to be particularly effective, because it directshigh levels of biological agents, such as antibodies or binding portionsthereof, probes, or ligands, to the bone marrow and lymph nodes whereprostate cancer metastases and metastases of many other cancerspredominate. Moreover, the methods of the present invention areparticularly well-suited for treating prostate cancer, because tumorsites for prostate cancer tend to be small in size and, therefore,easily destroyed by cytotoxic agents. Treatment in accordance with thepresent invention can be effectively monitored with clinical parameters,such as, in the case of prostate cancer, serum prostate specific antigenand/or pathological features of a patient's cancer, including stage,Gleason score, extracapsular, seminal, vesicle or perineural invasion,positive margins, involved lymph nodes, etc. Alternatively, theseparameters can be used to indicate when such treatment should beemployed.

[0098] Because the biological agents of the present invention bind toliving prostate cells, therapeutic methods for treating prostate cancerusing these biological agents are much more effective than those whichtarget lysed prostate cells. For the same reasons, diagnostic andimaging methods which determine the location of living normal, benignhyperplastic, or cancerous prostate epithelial cells (as well asvascular endothelial cells within cancers) are much improved byemploying the biological agents of the present invention. In addition,the ability to differentiate between living and dead prostate cells canbe advantageous, especially to monitor the effectiveness of a particulartreatment regimen.

[0099] Hybridomas E99, J415, J533, and J591 have been deposited pursuantto, and in satisfaction of, the requirements of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with the American Type Culture Collection(“A.T.C.C.” ) at 12301 Parklawn Drive, Rockville, Md. 20852. HybridomaE99 was deposited on May 2, 1996, and received A.T.C.C. DesignationNumber HB-12101. Hybridoma J415 was deposited on May 30, 1996, andreceived A.T.C.C. Designation Number HB-12109. Hybridomas J533 and J591were deposited on June 6, 1996, and received A.T.C.C. DesignationNumbers HB-12127 and HB-12126, respectively.

[0100] The present invention is further illustrated by the followingexamples.

EXAMPLES Example 1—Human Tissues

[0101] Fresh specimens of benign and malignant tissues were obtainedfrom the Department of Pathology of New York Hospital Cornell UniversityMedical Center (“NYH-CUMC”).

Example 2—Tissue Culture

[0102] Cultured cell lines of human cancers were obtained from theLaboratory of Urological Oncology of NYH-CUMC. The prostate cancer celllines PC-3 (Mickey, D. D., et al., “Characterization Of A Human ProstateAdenocarcinoma Cell Line (DU145) As A Monolayer Culture And As A SolidTumor In Athymic Mice,” Proc. Clin. Biol. Res., 37:67-84 (1980), whichis hereby incorporated by reference), DU-145 (Mickey, D. D., et al.,“Characterization Of A Human Prostate Adenocarcinoma Cell Line (DU145)As A Monolayer Culture And As A Solid Tumor In Athymic Mice,” Prog.Clin. Biol. Res., 37:67-84 (1980), which is hereby incorporated byreference), and LNCaP (Horoszewicz, J. S., et al., “LNCaP Model Of HumanProstatic Carcinoma,” Cancer Res., 43:1809-1818 (1983), which is herebyincorporated by reference) were obtained from the American Type CultureCollection (Rockville, Md.). Hybridomas were initially cloned inRPMI-1640 medium supplemented with 10% FCS, 0.1 mM nonessential aminoacids, 2mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml ofstreptomycin and HAT medium (GIBCO, Grand Island, N.Y.). Subclones werecultured in the same medium without aminopterin.

Example 3—Preparation of Mouse Monoclonal Antibodies

[0103] Female BALB/c mice were immunized intraperitoneally with LNCaP(6×10⁶ cells) three times at 2 week intervals. A final intraperitonealbooster immunization was administered with fresh prostate epithelialcells which had been grown in vitro. Three days later, spleen cells werefused with SP-2 mouse myeloma cells utilizing standard techniques (Ueda,R., et al., “Cell Surface Antigens Of Human Renal Cancer Defined ByMouse Monoclonal Antibodies: Identification Of Tissue-Specific KidneyGlycoproteins,” Proc. Natl. Acad. Sci. USA, 78:5122-5126 (1981), whichis hereby incorporated by reference). Supernatants of the resultingclones were screened by rosette and complement cytotoxicity assaysagainst viable LNCaP. Clones which were positive by these assays werescreened by immunochemistry vs normal kidney, colon, and prostate.Clones which were LNCap⁺/Nm1Kid⁻/colon⁻/prostate⁺ were selected andsubcloned 3 times by limiting dilution. The immunoglobulin class ofcultured supernatant from each clone was determined by immunodiffusionusing specified rabbit antisera (Calbiochem, San Diego, Calif.). mabswere purified using the MAPS-II kit (Bio-Rad, Richmond, Calif.)

Example 4—Biotinvlation of mabs

[0104] Purified mAbs were dialyzed in 0.1 M NaHCO₃ for 2 hours. One mlof mAb at 1 mg/ml was mixed with 0.1 ml of biotinamidocaproateN-hydroxysuccinamide ester (Sigma) in dimethylsulfoxide (1 mg/ml) andstirred for 4 hours at room temperature. Unbound biotin was removed bydialysis against phosphate buffered saline (“PBS”).

Example 5—Immunohistochemical Staining of Prostate Tissues

[0105] Cryostat sections of prostate tissues were placed inside rings ofFalcon 3034 plate covers (Becton-Dickenson, Lincoln Park, N.J.)previously coated with 0.45% gelatin solution as described in Marusich,M. F., “A Rapid Method For Processing Very Large Numbers Of TissueSections For Immunohistochemical Hybridoma Screening,” J. Immunol.Methods, 111:143-145 (1988), which is hereby incorporated by reference.Plates were stored at −80° C. Cryostat sections were fixed with 2%paraformaldehyde in PBS for 10 min at room temperature, and, afterwashing with PBS, endogenous peroxidase activity was blocked bytreatment with 0.3% hydrogen peroxide in PBS for 10 min at roomtemperature. After sections were incubated with 2% BSA in PBS for 20min, mAbs were added for 60 min at room temperature. Slides wereextensively washed with PBS and incubated with peroxidase-conjugatedrabbit anti-mouse Ig (DAKO Corp., Santa Barbara, Calif.) diluted 1:100in 10% normal human serum in PBS for 60 min at room temperature. After adiaminobenzidine reaction, sections were counterstained withhematoxylin.

Example 6—Serological Analysis

[0106] The anti-mouse immunoglobulin mixed hemadsorption assay wasperformed as described in Ueda, R., et al., “Cell Surface Antigens OfHuman Renal Cancer Defined By Mouse Monoclonal Antibodies:Identification Of Tissue-Specific Kidney Glycoproteins,” Proc. Natl.Acad. Sci. USA, 78:5122-5126 (1981), which is hereby incorporated byreference. To prepare the indicator cells, anti-mouse Ig (DAKO Corp.)was conjugated to type 0 human RBC using 0.01% chromium chloride.Serological assays were performed on cells previously plated in Terasakiplates (Nunc, Denmark). Antibodies were incubated with target cells atroom temperature for 1 hour. Target cells were then washed, andindicator cells added for 1 hour.

Example 7—Immunoprecipitation

[0107] LNCaP cells (2×10⁷) were biotinylated with biotin-NHSS (at finalconcentration of 5mM) for 30 minutes on ice. After washing, thebiotinylated cells were resuspended in 1 ml lysis buffer (20mM Tris/HClpH 8.0, 1 mM EDTA, 1 mM PMSF, 1% triton X-100) for 30 min on ice. Thesuspension was centrifuged at 1500×100 min at 4° C., and the supernatantwas centrifuged at 12,000 rpm×15 min at 4° C. The resulting lysate waspreabsorbed with rabbit or goat anti-mouse IgG-coated pansorbin for 1hour at 4° C. The pre-absorbed lysate was incubated with the mAbovernight at 4° C. Rabbit or goat anti-mouse Ig-coated agarose beadswere added for 2 hours at 4° C. and then washed. The beads wereresuspended in Tris-base/NaCl, added to sample buffer with2-mercaptoethanol, and boiled for 5 min. After centrifuging, thesupernatant was run on an SDS-PAGE 12% gel. The gel was transferred to anitrocellulose membrane which was blocked and stained withstraptavidin-peroxidase. The membrane was developed withdiaminobenzidine (“DAB”) Sequential immunoprecipitation was similarexcept that the lysate was initially pre-cleared with one mAb overnightat 40° C. A second mAb was then used to immunoprecipitate thepre-cleared lysate.

[0108] Approximately 2000 clones were screened, of which four cloneswere selected as described in Example 3, above. After subcloning,supernatants from the 4 hybridomas, E99, J415, J533, and J591, wereassayed by immunofluorescence against viable (i.e. unfixed) LNCaP,immunoprecipitation, and sequential immunoprecipitation to confirmreactivity to PSMA.

[0109] The immunofluorescence study using the LNCaP target cell(described originally in Horoszewicz, which is hereby incorporated byreference, to make the 7E11 antibody and the prototype cell line forexpression for PSMA) shows that E99 antibody binds to and renders viableLNCaP cells immunofluorescent. This is in contrast to the 7E11 antibody,which, as noted originally in Horoszewicz, which is hereby incorporatedby reference, gives only poor or no binding to viable LNCaP cells butexhibits strong binding once the cells are fixed (killed).

[0110] The reactivities of the four mAbs with normal human tissues wereexamined immunohistochemically; these results are presented in Table 3.TABLE 3 Reactivity of mAbs with human normal tissues by indirectimmunoperosidase staining E99 J415 J533 J591 Tissues (γ₃) (γ₁) (γ₁) (γ₁)Prostate*     Kidney ∘ ∘ ∘ ∘ Glomerulus ▪ ▪ ▪ ▪ Prox. Tubule ∘ ∘ ∘ ∘Ureter ∘ ∘ ∘ ∘ Bladder ∘ ∘ ∘ ∘ Testis ∘ ∘ ∘ ∘ Uterus ∘ ∘ ∘ ∘ Esophagus ∘∘ ∘ ∘ Small Intestine ∘ ∘ ∘ ∘ Stomach ∘ ∘ ∘ ∘ Colon ∘ ∘ ∘ ∘ Spleen ∘ ∘ ∘∘ Thyroid ∘ ∘ ∘ ∘ Lung ∘ ∘ ∘ ∘ Pancreas ∘ ∘ ∘ ∘ Liver ∘ ∘ ∘ ∘ * BPH 0-3⁺0-3⁺ 0-4⁺ 0-4⁺ * Prostate Cancer 0-3⁺ 0-3⁺ 0-4⁺ 0-4⁺ * LNCaP (scid) 3⁺3⁺ 4⁺ 4⁺ * LuCaP (scid) 0-2⁺ 0-2⁺ 0-3⁺ 0-3⁺

[0111] The above sequential immunoprecipitaion study showed that 7E11,E99, J415, J533, and J591 bind to the same molecule, i.e. PSMA.

Example 8—Western Blot Analysis

[0112] To confirm that antibodies E99, J415, J533, and J591 precipitatean identical band to the 7Ell antibody (i.e., PSMA), Western Blotanalyses were performed. Seminal plasma (400 μg/lane) or LNCaP lysatewere loaded into lanes of 12% SDS-PAGE gels. After electrophoresis, thegels are transferred to nitrocellulose membranes. The membranes wereblocked with 5% dry milk/Tris-buffered saline-tween 20 (“TBST”) for 60min at room temperature. After washing, the membranes were incubatedwith primary mAb for 60 min at room temperature. After repeat washing,the membranes were incubated with sheep anti-mouse-Ig-peroxidase 1/5000in 5% dry milk/TEST for 60 min at room temperature. After repeatwashing, the membranes were developed using a chemiluminescent tagdesignated “ECL” (Amersham Life Sciences, International, ArlingtonHeights, Ill.) according to the manufacturer's directions. The resultsof the Western Blot experiment are presented in Table 4. TABLE 4 Westernblot data Sample 7E11 E99 J415 J533 J591 Prostatic 100 KD 100 KD 100 KD100 KD 100 KD (seminal) band band band band band fluid LNCaP 100 KD &100 KD & 100 KD & 100 KD & 100 KD & cell lysate 200 KD 200 KD 200 KD 200KD 200 KD bands bands bands bands bands

Example 9—mAb Reactivity to External Domain of PSMA

[0113] To confirm cell surface (external) expression of the detectedPSMA, fresh, viable LNCaP cells were tested, without fixation, in vitro,by immunofluorescence. LNCaP cells were washed and incubated with mAbfor 1 hour at room temperature and then with a rabbit anti-mouseIg-fluorescein (DAKO Corp., Santa Barbara, Calif.). Wells were read witha fluorescent microscope. Negative control consisted of anisotype-matched irrelevant mAb, while an anti-class I MHC mAb served asa positive control.

[0114] Immunofluorescence and rosette assay results are presented inTable 5. TABLE 5 Comparison of 7E11 with new mAbs LNCaP viable cells7E11 E99 J415 J533 J591 Immunofluor- neg 3+ 3+ 4+ 4+ escence Rosetteneg + + + + assay LNCaP-fixed +++ ++++ +++ ++ +++

Example 10—Competition Studies

[0115] A competition study was carried out to determine whether J591,J533, E99, and J415 detected the same or different antigenic sites(epitopes) of the prostate specific membrane antigen molecule using thefollowing procedure.

[0116] Plates were coated with LNCaP cell line lysate as a source ofprostate specific membrane antigen and washed to remove unboundmaterial. “Cold” (unlabeled) monoclonal antibody was incubated on theplate for 1 hour at room temperature to allow binding to its antigenicsite. Subsequently, a second monoclonal antibody, labeled either withbiotin or ¹²⁵I, was added for an additional hour. Plates were washed toremove unbound material. The amount of the second monoclonal antibodybound to the prostate specific membrane antigen-coated plate wasdetermined either by avidin-alkaline phosphatase in an enzyme-linkedimmunoassay (in the case of biotin-labeled second monoclonal antibody)or by physically counting the well in a gamma counter (in the case of¹²⁵I-labeled second monoclonal antibody).

[0117] Controls consisted of using the same monoclonal antibody bothcold and labeled to define “100% competition” or using monoclonalantibody to a totally different molecule (e.g., monoclonal antibodyI-56, which detects inhibin, a prostate related protein different fromprostate specific membrane antigen) to define “0% competition”.

[0118] The results indicated that J591, J533, and E99 each interfere,compete, or block binding of one another but do not block binding ofJ415 and vice verse. 7E11/CYT356, known to bind PSMA at a different(intracellular) site, did not block any of J591, J533, E99, or J415.

[0119] Having pairs of monoclonal antibodies which bind to non-competingsites permits development of antibody sandwich assays for detectingsoluble antigens, such as solubilized prostate specific membrane antigenor fragment thereof, in, for example, body fluids. For example, theantigen (e.g., prostate specific membrane antigen or a fragment thereof)could be “captured” from body fluid with J591 and, in another step,detected by labeled J415.

[0120] In another setting, e.g. treatment, one could increase antibodybinding by using a combination of non-competing monoclonal antibodies.For example, assuming the non-competing sites are each represented onceon the prostate specific membrane antigen molecule, adding a combinationof J591 plus J415 would bind twice as many monoclonal antibody moleculesas either monoclonal antibody alone. Binding two non-competing antigenicbinding sites also can result in greater antigen cross-linking and,perhaps, increased internalization. Furthermore, since the two detectedsites are physically located on the same prostate specific membraneantigen molecule, the binding of two monoclonal antibody molecules tothat single prostate specific membrane antigen molecule puts the twomonoclonal antibody molecules in close proximity to each other, asetting which provides optimal drug-prodrug interaction. For example,monoclonal antibody J591 can be conjugated with an inactive pro-drug andJ415 can be conjugated with a pro-drug activator. Since prodrug andactivator would be bound in close proximity only at the site of prostatespecific membrane antigen-expressing cells (e.g., prostate cancercells), prodrug activation to the active form would occur only at thosesites.

Example 11—Microscopy

[0121] Confocal microscopy and immuno-electron microscopy demonstratedthat E99, J591, J533, and J415 are bound to the -cell membrane atclathrin-coated pits and then rapidly internalize into endosomes(cytoplasmic vesicles). FIGS. 1-4 are immuno-electron micrographs whichfollow the interaction of gold-labeled monoclonal antibody J591 with thecell surface as a function of time. In these figures, the location ofthe monoclonal antibody is indicated by the black dots.

[0122] Viable LNCaP cells were incubated with J591 for one hour at 4° C.The cells were washed and then held at 37° C. for 0, 5, 10, or 15minutes, after which time they were fixed and processed forimmuno-electron microscopy. FIG. 1 shows the cell prior to 37° C.incubation. J591 can be seen bound to the cell along the external aspectof the cell membrane. In this Figure, “M” denotes the cell'smitochondria, and “N” denotes its nucleus. FIG. 2 shows the cell afterincubation at 37° C. for 5 minutes. The arrow indicates formation of aclathrin-coated pit. In FIG. 3, which shows the cell after a 10 minute37° C. incubation, pinching off or endocytosis of the clathrin-coatedpit can be seen, as indicated by the arrow. FIG. 4 shows that, afterincubation at 37° C. for 15 minutes, monoclonal antibody J591 iscontained in endocytic vesicles within the cell, as indicated by thearrows. As can be seen in FIG. 5, after incubation at 37° C. for 15minutes, monoclonal antibody J591 is also contained within endosomes, asindicated by the arrows.

Example 12—Sequencing of the Variable Region of Monoclonal Antibody J591

[0123] Total RNA was prepared from 10⁷ murine hybridoma J591 cells. Asample of the conditioned medium from these cells was tested for bindingto the specific antigen for J591 on prostate cells. The conditionedmedium was positive by both ELISA and Western Blot for binding to theantigen.

[0124] VH and VK cDNA were prepared using reverse transcriptase andmouse κ constant region and mouse IgG constant region primers. The firststrand cDNAs were amplified by PCR using a variety of mouse signalsequence primers (6 for VH and 7 for VK). The amplified DNAs weregel-purified and cloned into the vector pT7Blue.

[0125] The VH and VK clones obtained were screened for correct insertsby PCR, and the DNA sequence of selected clones was determined by thedideoxy chain termination method.

[0126] Excluding the primer region (as the sequence of this depended onthe sequence of the primer that was used), all the VH clones obtainedgave identical sequence. This sequence was obtained from clones producedwith three different 5′ primers. One clone had one base pair changewithin the signal sequence, and one clone contained an aberrant PCRproduct. Using the sequencing strategy shown in FIG. 6, the nucleotidesequence for the heavy chain was obtained. It is designated SEQ. ID. No.1 and is presented in FIG. 7, along with the nucleotide sequence of thecorresponding reverse, non-coding strand (designated SEQ. ID. No. 2).

[0127] These sequences include part of the signal sequence and part ofthe constant region of the antibody. The corresponding deduced aminoacid sequences of J591 VH, designated SEQ. ID. No. 3, SEQ. ID. No. 4,and SEQ. ID. No. 5, are also shown in FIG. 7. The coding strand of theJ591 heavy chain's variable region (exclusive of signal sequence andconstant region components) has the following nucleotide sequence(designated SEQ. ID. No. 6):GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAGGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATACCATACACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAACATCAATCCTAACAATGGTGGTACCACCTACAATCAGAAGTTCGAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTAACATCTGAGGATTCTGCAGTCTATTATTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0128] The reverse, non-coding strand of the J591 heavy chain's variableregion (exclusive of signal sequence and constant region components) hasthe following nucleotide sequence (designated SEQ. ID. No. 7):TGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGTAGTCAAAGTTCCAACCAGCTGCACAATAATAGACTGCAGAATCCTCAGATGTTAGGCTGCGGAGCTCCATGTAGGCTGTACTGGAGGACTTGTCTACAGTCAATGTGGCCTTGTCCTCGAACTTCTGATTGTAGGTGGTACCACCATTGTTAGGATTGATGTTTCCAATCCACTCAAGGCTCTTTCCATGGCTCTGCTTCACCCAGTGTATGGTATATTCAGTGAATGTGTATCCAGAAGTCTTGCAGGATATCCTCACTGAAGTCCCAGGCTTCACCAGTTCAGGTCCAGACTGTTGCAGCTGGACCTC

[0129] The protein sequence corresponding to the J591 heavy chain'svariable region (exclusive of signal sequence and constant regioncomponents) has the following nucleotide sequence (designated SEQ. ID.No. 8): EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGW NFDYWGQGTTLTVSS

[0130] The J591 VH is in Mouse Heavy Chains Subgroup IIA (Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services (1991) (“Kabat”), which is hereby incorporatedby reference). The sequence of J591 VH is compared to the consensussequence for this subgroup in FIG. 8.

[0131] In contrast to the VH, more than one VK sequence was obtained.Out of the 15 VK clones examined, four gave the sequence of an aberrantmouse Igκ from the fusion partner (Carol et al., Molecular Immunology,25:991-995 (1988), which is hereby incorporated by reference). Theseclones originated from two specific 5′ primers. No further work was donewith these clones. Of the remaining clones, ten gave identicalnucleotide sequences, and one clone, VK17, gave an alternative VKsequence. The ten identical clones originated from three 5′ primers(different from the two that gave the aberrant sequence), one of whichalso produced VK17. The sequencing strategy that was employed is shownin FIG. 9.

[0132] The nucleic acid sequence of J591 VK corresponding to the tenidentical clones (designated SEQ. ID. No. 9) is presented in FIG. 10,along with the nucleic acid sequence of the corresponding reverse,non-coding strand (designated SEQ. ID. No. 10) and the deduced aminoacid sequences, which are designated SEQ. ID. No. 11, SEQ. ID. No. 12,and SEQ. ID. No. 13. These sequences include part of the signal sequenceand part of the constant region of the antibody. The coding strand ofthe J591 light (kappa) chain's variable region (exclusive of signalsequence and constant region components) corresponding to the tenidentical clones has the following nucleotide sequence (designated SEQ.ID. No. 14): AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA

[0133] The reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to the ten identical clones has the followingnucleotide sequence (designated SEQ. ID. No. 15):TTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGATAGCTGTAACCCTGTCCACAGTGATAATCTGCAAGGTCTTCAGCCTGCACACTGCTGATGGTCAGAGTGAAATCTGTTGCAGATCCACTGCCTGTGAAGCGATCGGGGACCCCAGTGTACCGGTTGGATGCCCCGTATATCAGCAGTTTAGGAGACTGCTCTGGTTTCTGTTGATACCAGGAAACATAAGTAACCACATTCTCACTGGCCTTGCAGGTCAAGGTGACCCTCTCTCCTACTGACATGGACATGGATTTGGG AGATTGGGTCATTACAATGTT

[0134] The protein sequence corresponding to the J591 light (kappa)chain's variable region (exclusive of signal sequence and constantregion components) corresponding to the ten identical clones has thefollowing nucleotide sequence (designated SEQ. ID. No. 16):NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGG GTKLEIK

[0135] The coding strand of the J591 light (kappa) chain's variableregion (exclusive of signal sequence and constant region components)corresponding to clone VK17 has the following nucleotide sequence(designated SEQ. ID. No. 17):GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAATGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCT GGGACCATGCTGGACCTGAAA

[0136] The reverse, non-coding strand of the J591 light (kappa) chain'svariable region (exclusive of signal sequence and constant regioncomponents) corresponding to clone VK17 has the following nucleotidesequence (designated SEQ. ID. No. 18):TTTCAGGTCCAGCATGGTCCCAGCACCGAACGTGAGAGGATAGCTGTTATATTGCTGACAGAAATAATCTGCCAAGTCTTCAGACTGAACATTAGTAATGGTGAGAGTGAAGTCTGTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACTCCAGTGTGCCGAGTGGATGCCCAATAAATCAGTAGTTTAGGAGATTGTCCTGGTTTCTGTTGATACCAGTCTACAGCAGTACCCACATCTTGACTGGCCTTACAGATGATGCTGACCCTGTCTCCTACTGATGTGGACATGAATTTGTG AGACTGGGTCATCACAATGTC

[0137] The protein sequence corresponding to the J591 light (kappa)chain's variable region (exclusive of signal sequence and constantregion components) corresponding to clone VK17 has the followingnucleotide sequence (designated SEQ. ID. No. 19):DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGA GTMLDLK

[0138] J591 VK is in the Mouse Kappa Chains Subgroup V (Kabat, which ishereby incorporated by reference). The sequence of J591 VK correspondingto the ten identical clones is compared to the consensus sequence forthe subgroup in FIG. 11.

[0139] Preferred J591's are those having heavy chain variable region DNAcoding strand sequences corresponding to SEQ. ID. No. 6 and non-codingstrand (reverse) sequences corresponding to SEQ. ID. No. 7. The heavychain variable region of J591 preferably has an amino acid sequencecorresponding to SEQ. ID. No. 8. The light chain variable region of J591preferably has a DNA coding strand sequence corresponding to SEQ. ID.No. 17, a DNA non-coding strand (reverse) sequence corresponding to SEQ.ID. No. 18, and a amino acid sequence corresponding to SEQ. ID. No. 19.

Example 13—Immunohistochemical Staining of Normal and Cancer Tissues

[0140] Cancer tissues from 23 carcinomas were pre-cooled in liquidnitrogen, snap-frozen in OCT compound (Miles, Elkhart, Ind.) on dry ice,and stored at −80° C. Cryostat tissue sections (5 μm) were fixed in coldacetone (4° C.) for 10 minutes. mAbs (5 μg/ml or hybridoma supernatants)were incubated for 1 hour at room temperature. Antibody binding wasdetected using rabbit anti-mouse Ig-peroxidase (Dako, Carpinteria,Calif.) as a secondary antibody and DAB (Sigma, St. Louis, Mo.) aschromogen. Isotype-matched irrelevant antibody was used as negativecontrol.

[0141] mAbs J591, J533, J415, and E99 reacted strongly with vascularendothelia in all 23 carcinomas studied, including 9/9 renal, 5/5urothelial, 6/6 colon, 1/1 lung, and 1/1 breast carcinomas, and 1/1metastatic adenocarcinoma to the liver. FIGS. 2A-2F, respectively, showthe immunohistochemical reactivity of mAb J591 to neovasculature ofrenal, urothelial, colon, lung, and breast carcinomas, and metastaticadenocarcinoma to the liver.

[0142] Although the invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose and variations can be made by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1 21 1 391 DNA Mus sp. 1 tctcctgtca ggaactgcag gtgtcctctc tgaggtccagctgcaacagt ctggacctga 60 actggtgaag cctgggactt cagtgaggat atcctgcaagacttctggat acacattcac 120 tgaatatacc atacactggg tgaagcagag ccatggaaagagccttgagt ggattggaaa 180 catcaatcct aacaatggtg gtaccaccta caatcagaagttcgaggaca aggccacatt 240 gactgtagac aagtcctcca gtacagccta catggagctccgcagcctaa catctgagga 300 ttctgcagtc tattattgtg cagctggttg gaactttgactactggggcc aaggcaccac 360 tctcacagtc tcctcagcca aaacgacacc c 391 2 391DNA Mus sp. 2 gggtgtcgtt ttggctgagg agactgtgag agtggtgcct tggccccagtagtcaaagtt 60 ccaaccagct gcacaataat agactgcaga atcctcagat gttaggctgcggagctccat 120 gtaggctgta ctggaggact tgtctacagt caatgtggcc ttgtcctcgaacttctgatt 180 gtaggtggta ccaccattgt taggattgat gtttccaatc cactcaaggctctttccatg 240 gctctgcttc acccagtgta tggtatattc agtgaatgtg tatccagaagtcttgcagga 300 tatcctcact gaagtcccag gcttcaccag ttcaggtcca gactgttgcagctggacctc 360 agagaggaca cctgcagttc ctagcaggag a 391 3 123 PRT Mus sp.3 Ser Pro Val Arg Asn Cys Arg Cys Pro Leu Gly Pro Ala Ala Thr Val 1 5 1015 Trp Thr Thr Gly Glu Ala Trp Asp Phe Ser Glu Asp Ile Leu Gln Asp 20 2530 Phe Trp Ile His Ile His Ile Tyr His Thr Leu Gly Glu Ala Glu Pro 35 4045 Trp Lys Glu Pro Val Asp Trp Lys His Gln Ser Gln Trp Trp Tyr His 50 5560 Leu Gln Ser Glu Val Arg Gly Gln Gly His Ile Asp Cys Arg Gln Val 65 7075 80 Leu Gln Tyr Ser Leu His Gly Ala Pro Gln Pro Asn Ile Gly Phe Cys 8590 95 Ser Leu Leu Leu Cys Ser Trp Leu Glu Leu Leu Leu Gly Pro Arg His100 105 110 His Ser His Ser Leu Leu Ser Gln Asn Asp Thr 115 120 4 130PRT Mus sp. 4 Leu Leu Ser Gly Thr Ala Gly Val Leu Ser Glu Val Gln LeuGln Gln 1 5 10 15 Ser Gly Pro Glu Leu Val Lys Pro Gly Thr Ser Val ArgIle Ser Cys 20 25 30 Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile HisTrp Val Lys 35 40 45 Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asn IleAsn Pro Asn 50 55 60 Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp LysAla Thr Leu 65 70 75 80 Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met GluLeu Arg Ser Leu 85 90 95 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala AlaGly Trp Asn Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr ValSer Ser Ala Lys Thr 115 120 125 Thr Pro 130 5 125 PRT Mus sp. 5 Leu SerCys Gln Glu Leu Gln Val Ser Ser Leu Arg Ser Ser Cys Asn 1 5 10 15 SerLeu Asp Leu Asn Trp Ser Leu Gly Leu Gln Gly Tyr Pro Ala Arg 20 25 30 LeuLeu Asp Thr His Ser Leu Asn Ile Pro Tyr Thr Gly Ser Arg Ala 35 40 45 MetGlu Arg Ala Leu Ser Gly Leu Glu Thr Ser Ile Leu Thr Met Val 50 55 60 ValPro Pro Thr Ile Arg Ser Ser Arg Thr Arg Pro His Leu Thr Ser 65 70 75 80Pro Pro Val Gln Pro Thr Trp Ser Ser Ala Ala His Leu Arg Ile Leu 85 90 95Gln Ser Ile Ile Val Gln Leu Val Gly Thr Leu Thr Thr Gly Ala Lys 100 105110 Ala Pro Leu Ser Gln Pro Ser Gln Pro Lys Arg His Pro 115 120 125 6345 DNA Mus sp. 6 gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctgggacttcagtgaggata 60 tcctgcaaga cttctggata cacattcact gaatatacca tacactgggtgaagcagagc 120 catggaaaga gccttgagtg gattggaaac atcaatccta acaatggtggtaccacctac 180 aatcagaagt tcgaggacaa ggccacattg actgtagaca agtcctccagtacagcctac 240 atggagctcc gcagcctaac atctgaggat tctgcagtct attattgtgcagctggttgg 300 aactttgact actggggcca aggcaccact ctcacagtct cctca 345 7345 DNA Mus sp. 7 tgaggagact gtgagagtgg tgccttggcc ccagtagtca aagttccaaccagctgcaca 60 ataatagact gcagaatcct cagatgttag gctgcggagc tccatgtaggctgtactgga 120 ggacttgtct acagtcaatg tggccttgtc ctcgaacttc tgattgtaggtggtaccacc 180 attgttagga ttgatgtttc caatccactc aaggctcttt ccatggctctgcttcaccca 240 gtgtatggta tattcagtga atgtgtatcc agaagtcttg caggatatcctcactgaagt 300 cccaggcttc accagttcag gtccagactg ttgcagctgg acctc 345 8115 PRT Mus sp. 8 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val LysPro Gly Thr 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr ThrPhe Thr Glu Tyr 20 25 30 Thr Ile His Trp Val Lys Gln Ser His Gly Lys SerLeu Glu Trp Ile 35 40 45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr TyrAsn Gln Lys Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser SerSer Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp SerAla Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly GlnGly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 9 363 DNA Mus sp. 9ttatatggag ctgatgggaa cattgtaatg acccaatctc ccaaatccat gtccatgtca 60gtaggagaga gggtcacctt gacctgcaag gccagtgaga atgtggttac ttatgtttcc 120tggtatcaac agaaaccaga gcagtctcct aaactgctga tatacggggc atccaaccgg 180tacactgggg tccccgatcg cttcacaggc agtggatctg caacagattt cactctgacc 240atcagcagtg tgcaggctga agaccttgca gattatcact gtggacaggg ttacagctat 300ccgtacacgt tcggaggggg gaccaagctg gaaataaaac gggctgatgc tgcaccaact 360gta 363 10 363 DNA Mus sp. 10 tacagttggt gcagcatcag cccgttttatttccagcttg gtcccccctc cgaacgtgta 60 cggatagctg taaccctgtc cacagtgataatctgcaagg tcttcagcct gcacactgct 120 gatggtcaga gtgaaatctg ttgcagatccactgcctgtg aagcgatcgg ggaccccagt 180 gtaccggttg gatgccccgt atatcagcagtttaggagac tgctctggtt tctgttgata 240 ccaggaaaca taagtaacca cattctcactggccttgcag gtcaaggtga ccctctctcc 300 tactgacatg gacatggatt tgggagattgggtcattaca atgttcccat cagctccata 360 taa 363 11 121 PRT Mus sp. 11 LeuTyr Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro Lys Ser 1 5 10 15Met Ser Met Ser Val Gly Glu Arg Val Thr Leu Thr Cys Lys Ala Ser 20 25 30Glu Asn Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr 65 70 7580 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln 85 9095 Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val 115 120 12 114 PRT Mus sp.12 Tyr Met Glu Leu Met Gly Thr Leu Pro Asn Leu Pro Asn Pro Cys Pro 1 510 15 Cys Gln Glu Arg Gly Ser Pro Pro Ala Arg Pro Val Arg Met Trp Leu 2025 30 Leu Met Phe Pro Gly Ile Asn Arg Asn Gln Ser Ser Leu Leu Asn Cys 3540 45 Tyr Thr Gly His Pro Thr Gly Thr Leu Gly Ser Pro Ile Ala Ser Gln 5055 60 Ala Val Asp Leu Gln Gln Ile Ser Leu Pro Ser Ala Val Cys Arg Leu 6570 75 80 Lys Thr Leu Gln Ile Ile Thr Val Asp Arg Val Thr Ala Ile Arg Thr85 90 95 Arg Ser Glu Gly Gly Pro Ser Trp Lys Asn Gly Leu Met Leu His Gln100 105 110 Leu Tyr 13 116 PRT Mus sp. 13 Ile Ile Trp Ser Trp Glu HisCys Asn Asp Pro Ile Ser Gln Ile His 1 5 10 15 Val His Val Ser Arg ArgGlu Gly His Leu Asp Leu Gln Gly Gln Glu 20 25 30 Cys Gly Tyr Leu Cys PheLeu Val Ser Thr Glu Thr Arg Ala Val Ser 35 40 45 Thr Ala Asp Ile Arg GlyIle Gln Pro Val His Trp Gly Pro Arg Ser 50 55 60 Leu His Arg Gln Trp IleCys Asn Arg Phe His Ser Asp His Gln Gln 65 70 75 80 Cys Ala Gly Arg ProCys Arg Leu Ser Leu Trp Thr Gly Leu Gln Leu 85 90 95 Ser Val His Val ArgArg Gly Asp Gln Ala Gly Asn Lys Thr Gly Cys 100 105 110 Cys Thr Asn Cys115 14 321 DNA Mus sp. 14 aacattgtaa tgacccaatc tcccaaatcc atgtccatgtcagtaggaga gagggtcacc 60 ttgacctgca aggccagtga gaatgtggtt acttatgtttcctggtatca acagaaacca 120 gagcagtctc ctaaactgct gatatacggg gcatccaaccggtacactgg ggtccccgat 180 cgcttcacag gcagtggatc tgcaacagat ttcactctgaccatcagcag tgtgcaggct 240 gaagaccttg cagattatca ctgtggacag ggttacagctatccgtacac gttcggaggg 300 gggaccaagc tggaaataaa a 321 15 321 DNA Mus sp.15 ttttatttcc agcttggtcc cccctccgaa cgtgtacgga tagctgtaac cctgtccaca 60gtgataatct gcaaggtctt cagcctgcac actgctgatg gtcagagtga aatctgttgc 120agatccactg cctgtgaagc gatcggggac cccagtgtac cggttggatg ccccgtatat 180cagcagttta ggagactgct ctggtttctg ttgataccag gaaacataag taaccacatt 240ctcactggcc ttgcaggtca aggtgaccct ctctcctact gacatggaca tggatttggg 300agattgggtc attacaatgt t 321 16 107 PRT Mus sp. 16 Asn Ile Val Met ThrGln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val ThrLeu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp TyrGln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala SerAsn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser AlaThr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp LeuAla Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys 100 105 17 321 DNA Mus sp. 17 gacattgtgatgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcatctgtaaggccagtca agatgtgggt actgctgtag actggtatca acagaaacca 120 ggacaatctcctaaactact gatttattgg gcatccactc ggcacactgg agtccctgat 180 cgcttcacaggcagtggatc tgggacagac ttcactctca ccattactaa tgttcagtct 240 gaagacttggcagattattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300 gggaccatgctggacctgaa a 321 18 321 DNA Mus sp. 18 tttcaggtcc agcatggtcc cagcaccgaacgtgagagga tagctgttat attgctgaca 60 gaaataatct gccaagtctt cagactgaacattagtaatg gtgagagtga agtctgtccc 120 agatccactg cctgtgaagc gatcagggactccagtgtgc cgagtggatg cccaataaat 180 cagtagttta ggagattgtc ctggtttctgttgataccag tctacagcag tacccacatc 240 ttgactggcc ttacagatga tgctgaccctgtctcctact gatgtggaca tgaatttgtg 300 agactgggtc atcacaatgt c 321 19 107PRT Mus sp. 19 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr SerVal Gly 1 5 10 15 Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp ValGly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro LysLeu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp ArgPhe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr AsnVal Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr AsnSer Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys 100105 20 125 PRT Mus sp. 20 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu LeuVal Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser GlyTyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Asn Trp Val Lys Gln Ser ProGly Lys Ser Leu Glu Trp 35 40 45 Ile Gly Asp Ile Asn Pro Gly Asn Gly GlyThr Ser Tyr Asn Gln Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Val AspLys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Tyr Ser Ser SerTyr Met Ala Tyr Tyr Ala Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser 115 120 125 21 109 PRT Mus sp. 21 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Asp Ile Ser Asn 20 25 30 Tyr Leu AsnTrp Tyr Gln Gln Lys Pro Gly Gly Ser Pro Lys Leu Leu 35 40 45 Ile Tyr TyrAla Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser GlySer Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu 65 70 75 80 Gln GluAsp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95 Pro ArgThr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

What is claimed:
 1. A method of ablating or killing cancerous cellscomprising: providing a biological agent which, when contacted with anextracellular domain of prostate specific membrane antigen, binds to theextracellular domain of prostate specific membrane antigen andcontacting vascular endothelial cells proximate to the cancerous cellswith the biological agent under conditions effective to permit bothbinding of the biological agent to the vascular endothelial cellsproximate to the cancerous cells and ablating or killing of thecancerous cells.
 2. A method according to claim 1, wherein thebiological agent kills or ablates the vascular endothelial cellsproximate to the cancerous cells, thereby killing or ablating thecancerous cells by reducing blood flow thereto.
 3. A method according toclaim 1, wherein the cancerous cells are renal cancerous cells,urothelial cancerous cells, colon cancerous cells, rectal cancerouscells, lung cancerous cells, breast cancerous cells, or cancerous cellsof metastatic adenocarcinoma to the liver.
 4. A method according toclaim 1, wherein the biological agent is an antibody or binding portionthereof, probe, or ligand.
 5. A method according to claim 1, wherein thebiological agent, when contacted with an extracellular domain ofprostate specific membrane antigen, is internalized with the prostatespecific membrane antigen.
 6. A method according to claim 1, whereinsaid contacting is carried out in a living mammal and comprises:administering the biological agent to the mammal under conditionseffective to permit both binding of the biological agent to vascularendothelial cells proximate to the cancerous cells and killing of thecancerous cells.
 7. A method according to claim 6, wherein saidadministering is carried out orally, parenterally, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, or by application tomucous membranes.
 8. A method according to claim 4, wherein an antibodyis used in carrying out said method, the antibody being selected fromthe group consisting of a monoclonal antibody and a polyclonal antibody.9. A method according to claim 8, wherein the antibody is selected fromthe group consisting of an E99, a J415, a J533, and a J591 monoclonalantibody.
 10. A method according to claim 8, wherein the antibody is amonoclonal antibody produced by a hybridoma cell line having an ATCCAccession Number selected from the group consisting of HB-12101,HB-12109, HB-12127, and HB-12126.
 11. A method according to claim 4,wherein a binding portion of an antibody is used in carrying out saidmethod, the binding portion being selected from the group consisting ofan Fab fragment, an F(ab′)₂ fragment, and an Fv fragment.
 12. A methodaccording to claim 4, wherein the probe or ligand is used in carryingout said method.
 13. A method according to claim 1, wherein thebiological agent is bound to a substance effective to kill or ablate thecancerous cells upon binding of the biological agent to vascularendothelial cells proximate to the cancerous cells.
 14. A methodaccording to claim 13, wherein the substance effective to kill or ablatethe cancerous cells is a cytotoxic drug.
 15. A method according to claim14, wherein the cytotoxic drug is selected from the group consisting oftherapeutic drug, a compound emitting radiation, molecules of plant,fungal, or bacterial origin, biological proteins, and mixtures thereof.16. A method according to claim 4, wherein the antibody is effective toinitiate an endogenous host immune function.
 17. A method according toclaim 16, wherein the endogenous host immune function iscomplement-mediated cellular cytoxicity.
 18. A method according to claim16, wherein the endogenous host immune function is antibody-dependentcellular cytoxicity.
 19. A method according to claim 1, wherein thebiological agent is in a composition further comprising aphysiologically acceptable carrier, excipient, or stabilizer.
 20. Amethod according to claim 1, wherein the biological agent is in acomposition further comprising a pharmaceutically acceptable carrier,excipient, or stabilizer.
 21. A method of detecting cancerous tissue ina biological sample comprising: providing an biological agent which,when contacted with an extracellular domain of prostate specificmembrane antigen, binds to the extracellular domain of prostate specificmembrane antigen, wherein the biological agent is bound to a labeleffective to permit detection of vascular endothelial cells proximate toor within the cancerous tissue upon binding of the biological agent tothe vascular endothelial cells proximate to or within the canceroustissue; contacting the biological sample with the biological agenthaving a label under conditions effective to permit binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue in the biological sample; and detecting apresence of any cancerous tissue in the biological sample by detectingthe label.
 22. A method according to claim 21, wherein the canceroustissue is renal cancerous tissue, urothelial cancerous tissue, coloncancerous tissue, rectal cancerous tissue, lung cancerous tissue, breastcancerous tissue, or cancerous tissue of metastatic adenocarcinoma tothe liver.
 23. A method according to claim 21, wherein the biologicalagent is an antibody or binding portion thereof, probe, or ligand.
 24. Amethod according to claim 21, wherein the biological agent, whencontacted with an extracellular domain of prostate specific membraneantigen, is internalized with the prostate specific membrane antigen.25. A method according to claim 21, wherein said contacting is carriedout in a living mammal and comprises: administering the biological agentto the mammal under conditions effective to permit binding of thebiological agent to the vascular endothelial cells proximate to orwithin the cancerous tissue in the biological sample.
 26. A methodaccording to claim 25, wherein the label is a short-range radiationemitter.
 27. A method according to claim 25, wherein said administeringis carried out orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intraversal instillation, byintracavitary or intravesical instillation, intraocularly,intraarterially, intralesionally, or by application to mucous membranes.28. A method according to claim 23, wherein an antibody is used incarrying out said method, said antibody being selected from the groupconsisting of a monoclonal antibody and a polyclonal antibody.
 29. Amethod according to claim 28, wherein the antibody is selected from thegroup consisting of an E99, a J415, a J533, and a J591 monoclonalantibody.
 30. A method according to claim 28, wherein the antibody is amonoclonal antibody produced by a hybridoma cell line having an ATCCAccession Number selected from the group consisting of HB-12101,HB-12109, HB-12127, and HB-12126.
 31. A method according to claim 23,wherein a binding portion of an antibody is used in carrying out saidmethod, the binding portion being selected from the group Consisting ofan Fab fragment, an F(ab′)₂ fragment, and an Fv fragment.
 32. A methodaccording to claim 23, wherein a probe or ligand is used in carrying outsaid method.
 33. A method according to claim 21, wherein the label isselected from the group consisting of a fluorescent label, a radioactivelabel, a nuclear magnetic resonance active label, a luminescent label,and a chromophore label.
 34. A method according to claim 21, wherein thebiological agent is in a composition further comprising aphysiologically acceptable carrier, excipient, or stabilizer.
 35. Amethod according to claim 21, wherein the biological agent is in acomposition further comprising a pharmaceutically acceptable carrier,excipient, or stabilizer.
 36. A method according to claim 21, whereinsaid contacting is carried out in a sample of serum or urine.
 37. Amethod according to claim 21, wherein said contacting is carried out ina tissue biopsy sample.